Central nervous system (CNS) dysfunction is often found in association with HIV-1 infection, yet the pathogenic mechanisms of AIDS-related CNS disorders are poorly understood. Massive and diverse neuropathological findings in HIV-1 infected individuals that affect all cell types in the CNS with restricted infection of these cells suggests that HIV-1 associated neuropathogenesis involves complex regulatory pathways that elicit direct and indirect effects of both viral and cellular factors. A high titers of HIV-1 are found predominantly in microglial cells and macrophages of the brain, it is suspected that these cells play an important role in inducing disease both directly by releasing virus, and indirectly by secreting viral and cellular factors that have an impact upon other CNS cells. Also, astroglial cells received special attention as the viral genome has been repeatedly detected in this cell type and in cell- culture system they support, albeit to a lesser extent, HIV-1 replication. Work from several laboratories has inducted that the secretion of the HIV-regulatory protein, Tat, and/or Tat-induced cytokines and immunomodulators by infected cells could affect neighboring uninfected cells and alter the expression of important genes. In support of this concept, our recent work has demonstrated that Tat has the ability to deregulate astrocyctic cell proliferation by elongating the G1 phase of the cell cycle and arresting cells at the G1/S boundary. Studies have revealed that Tat can alter the programmed expression of several key cell cycle regulatory proteins, including cyclins and their associated kinases. These alterations, which may affect the phosphorylation status of their downstream target protein, including pRb, can incapacitate E2F-1, a transcription factor whose activity is essential for S-phase entry, by maintaining E2F-1 in complex with hypophosphorylated pRb. On the other hand, we have demonstrated that active E2F-1, free from pRb, has the ability to suppress transcription of the HIV-1 LTR by utilizing a sequence spanning the HIV-1 kB activation motif. Thus, our transcription of the HIV-1 LTR by utilizing a sequence spanning the HIV-1 kB activation motif. Thus, our hypothesis is that HIV-1 Tat and its responsive cytokines, i.e. TNFalpha and TGFbeta, which have an impact upon the cell cycle regulatory apparatus, deregulate the cascade of events which are important for normal cell function. This, in turn, affects viral gene expression and replication since E2F-1, which suppresses HIV-1 gene transcription, remains sequestered by complexation with its cellular partner, pRb. In this proposal, we intend to study the interplay between Tat and Tat-induced cytokines, including TNFalpha and TGFbeta-1, with cell cycle regulatory proteins from human primary microglia and astrocytes by: (i) treating cells with HIV-1 Tat and the relevant cytokines at various stages of the cell cycle and examining key parameters which are important for control of the cell cycle, and (ii) expressing E2F-1 while investigating expression and replication of the HIV-1 viral genome before and after activation of NFkappaB and assess the in vivo association of E2F-1 and NFkappaB with their common motif on the HIV-1 LTR and with each other in these cells. Furthermore, in collaboration with the Neuropathology and Tissue Culture Core (Core A), expression and interaction on viral and relevant cellular proteins in clinical specimens will be investigated.